https://phaidra.vetmeduni.ac.at/o:4833 Glycoproteomic and Single-Protein Glycomic Analyses Reveal Zwitterionic N-Glycans on Natural and Recombinant Proteins Derived From Insect Cells Yan, Shi Shi Yan 0000-0003-4949-9140 University of Veterinary Medicine Vienna Vanbeselaere, Jorick Jorick Vanbeselaere Ives, Callum Callum Ives Stenitzer, David David Stenitzer Nuschy, Lena Lena Nuschy Woels, Florian Florian Woels Paschinger, Katharina Katharina Paschinger Fadda, Elisa Elisa Fadda Stadlmann, Johannes Johannes Stadlmann Wilson, Iain Iain Wilson American Society for Biochemistry and Molecular Biology journal article open access eng Insect cells are a convenient cell factory to produce recombinant glycoproteins. Their glycosylation potential is believed to be simple, needing primarily addition of glycosyltransferases to humanize the recombinant products. In this study, the native glycoproteome of Spodoptera frugiperda Sf9 and Trichoplusia ni High Five cells, examined using an LC-MS/MS approach, revealed not only which proteins are N-glycosylated but also indicated that the N-glycomes contain novel glucuronylated and phosphorylcholine-modified glycans, in addition to typical oligomannosidic and fucosylated structures. These data were corroborated by a parallel MALDI-TOF MS/MS analysis of N-glycosidase-released oligosaccharides. Molecular modeling analysis of one endogenous Sf9 glycoprotein correlated the occurrence of complex and oligomannosidic N-glycans with the accessibility of the occupied N-glycosylation sites. Further, we showed that the N-glycans of influenza hemagglutinins and SARS-CoV-2 spike glycoprotein produced in Spodoptera cells possess a number of glycan structures modified with phosphorylcholine, but core difucosylation was minimal; in contrast, the Trichoplusia-produced hemagglutinin had only traces of the former type, while the latter was dominant. Detection of phosphorylcholine on these glycoproteins correlated with binding to human C-reactive protein. In conclusion, not just oligomannosidic or truncated paucimannosidic N-glycans, but structures with immunogenic features occur on both natural and recombinant glycoproteins derived from insect cell lines. Baculovirus Fucose Glucuronic Acid Insect Cells N-glycans Phosphorylcholine http://creativecommons.org/licenses/by/4.0/ https://phaidra.vetmeduni.ac.at/api/object/o:4833/download 10.1016/j.mcpro.2025.100981 https://phaidra.vetmeduni.ac.at/o:605 application/pdf Molecular & Cellular Proteomics issn:1535-9484 VoR Molecular & Cellular Proteomics 24 6 4.81 MB 2025